The invention relates to the use of signal processing methods in order to acheive higher signal to noise ratios, to increase the detection limits of target analytes. These techniques include the monitoring of the output signal at higher harmonic frequencies.
There are a number of assays and sensors for the detection of the presence and/or concentration of specific substances in fluids and gases. Many of these rely on specific ligand/antiligand reactions as the mechanism of detection. That is, pairs of substances (i.e. the binding pairs or ligand/antiligands) are known to bind to each other, while binding little or not at all to other substances. This has been the focus of a number of techniques that utilize these binding pairs for the detection of the complexes. These generally are done by labelling one component of the complex in some way, so as to make the entire complex detectable, using, for example, radioisotopes, fluorescent and other optically active molecules, enzymes, etc.
Other assays rely on electronic signals for detection. Of particular interest are biosensors. At least two types of biosensors are known; enzyme-based or metabolic biosensors and binding or bioaffinity sensors. See for example U.S. Pat. Nos. 4,713,347; 5,192,507; 4,920,047; 3,873,267; and references disclosed therein. While some of these known sensors use alternating current (AC) techniques, these techniques are generally limited to the detection of differences in bulk (or dielectric) impedance.
Similarly, electronic detection of nucleic acids using electrodes is also known; see for example U.S. Pat. Nos. 5,591,578; 5,824,473; 5,705,348; 5,780,234 and 5,770,369; U.S. Ser. Nos. 08/873,598 08/911,589; and WO 98/20162; PCT/US98/12430; PCT/US98/12082; PCT/US99/10104; PCT/US99/01705, and PCT/US99/01703.
In the area of electrochemical sensing, there are a number of electrochemical techniques that have been employed. Traditionally, electrochemical methods generally improve their signal to noise ratios by discriminating the faradaic signal form the background components in the time domain through the application of pulsed waveforms, i.e. differnetail pulse polarography and square wave voltammetry. Pulse methods are able to discriminate the faradaic current from the changing current in the time domain. Changing currents decay much more rapidly than faradaic current, i.e. exponentially as compared the inverse square root. Similarly, modulation techniques have also be used to improve signal to noise ratios; these methods utilize the imposition of a modulated carrier wave (sine wave) on the signal.
The frequency domain has only bene used in a few electrochemical techniques to enhance the signal to noise ratio. In AC voltammetry, a potential ramp is applied to the electrode, and a small amplitude sine wave is superimposed on the linear ramp. However, the use of large amplitude sinosoidal voltammetry in conjunction with the detection at higher harmonic frequencies using Fourier transforms has proven to be a useful method. See U.S. Pat. No. 5,650,061; Singhai et al., Anal. Chem. 69:1552 (1997); Singhal and Kuhr, Anal. Chem. 69:4828 (1997); Singhal et al., Anal. Chem. 69:3553 (1997); and Dontha et al., Anal. Chem. 69:2619 (1997), all of which are expressly incorporated by reference herein.
However, further methods are still needed to exploit signal processing advantages in detecting biomolecules such as target analytes.
Accordingly, it is an object of the invention to provide novel methods and compositions for the detection analytes using AC techniques. These techniques find particular use in array formats, and for the detection of biomolecules such as nucleic acids and proteins.
In accordance with the objects outlined above, the present invention provides compositions and methods useful in detecting target analytes in a sample. The methods comprise providing an array comprising a plurality of electrodes, at least one of which comprises an assay complex. The assay complex comprises a capture binding ligand covalently attached to the electrode, a target analyte, and an electron transfer moiety. At least a first input signal is applied to the assay complex and an output signal is received. The output signal is then processed to detect the presence of said target analytes. Preferred embodiments utilize a plurality of assay complexes each attached to a different cell or pad of the array.